InteractiveFly: GeneBrief

Claspin: Biological Overview | References


Gene name - Claspin

Synonyms -

Cytological map position - 64B1-64B1

Function - adaptor protein

Keywords - ATR-Chk1 checkpoint pathway - required for the cell cycle checkpoint induced at midblastula transition - required for initiation and efficient progression of the replication forks during DNA replication

Symbol - Claspin

FlyBase ID: FBgn0052251

Genetic map position - chr3L:4,313,490-4,318,645

NCBI classification - Elongation factor Tu GTP binding domain

Cellular location - nuclear



NCBI link: EntrezGene
BIOLOGICAL OVERVIEW

Claspin and TopBP1 are checkpoint mediators that are required for the phosphorylation of Chk1 by ATR to maintain genomic stability. This study investigated the functions of Drosophila Claspin and mus101 (TopBP1 ortholog) during chorion (eggshell component) gene amplification, which occurs in follicle cells in the absence of global genomic DNA replication. Unlike Drosophila mei-41 (ATR ortholog) mutant embryos, Claspin and mus101 mutant embryos showed severe eggshell defects resulting from defects in chorion gene amplification. EdU incorporation assay during initiation and elongation stages revealed that Claspin and mus101 were required for initiation, while only Claspin had a major role in the efficient progression of the replication forks. Claspin proteins were enriched in the amplification foci both in the initiation and elongation stage-follicle cell nuclei in a mei-41-independent manner. The focal localization of ORC2, a component of the origin recognition complex, was not significantly affected in the Claspin mutant, whereas it was reduced in the mus101 mutant. It is concluded that Drosophila Claspin plays a major role in the initiation and elongation stages of chorion gene amplification by localizing to the amplification foci in a mei-41-independent manner. Drosophila mus101 is also involved in chorion gene amplification, mostly functioning in initiation, rather than elongation (Choi, 2017).

To maintain genomic stability, the ATR and Chk1 checkpoint kinases play major roles in the DNA damage checkpoint response, which is induced by various types of DNA damage, including DNA replication stress. DNA replication stress activates these checkpoint genes, leading to inhibition of mitotic entry and stabilization of the replication fork to prevent fork collapse. Claspin and TopBP1 are checkpoint mediators that enhance ATR activity. In addition to their checkpoint functions, Chk1, Claspin, and TopBP1 are involved in normal DNA replication (Petermann, 2008). The importance of the ATR, Chk1, Claspin (Yang, 2016), and TopBP1 genes during normal cell cycle progression is underscored by the embryonic lethality that results from mutations in these genes in mice. Drosophila contains the mei-41, Claspin, mus101, and grp genes, which are orthologs of ATR, Claspin, TopBP1, and Chk1, respectively. Studies of Drosophila Claspin mutants have demonstrated the involvement of Claspin in a replication stress-induced checkpoint during the midblastula transition (Lee, 2012), after hydroxyurea feeding (Lee, 2012), and in response to defective tRNA processing (Molla-Herman, 2015). Although the functions of Claspin during the checkpoint response have been extensively studied, its role during normal development is not well understood (Choi, 2017).

In the Drosophila ovary, somatic follicle cells encircle 16 germline cells, including the oocyte, and various cell cycle events occur in these follicle cells depending on their developmental stages. In addition to mitotic division, atypical cell cycle events, such as endoreplication and specific gene amplification in the absence of genomic replication, occur in somatic follicle cells during Drosophila oogenesis. During early development up to stage 6, follicle cells increase in number by undergoing mitotic divisions. Between stages 7 and 9, these cells endocycle by alternating between the S and gap phases. At stage 10, they cease genomic replication, and re-replication occurs from specific replication origins to amplify up to 60 copies of the chorion gene. The initiation and elongation stages of chorion gene replication occur during separate developmental stages of follicle cells; initiation occurs during stages 10B and 11, whereas only elongation from existing replication forks takes place during stages 12 and 13 (Choi, 2017).

Chorion is a major component of the eggshell and defects in chorion gene amplification result in a thin eggshell phenotype. Re-replication of the chorion gene induces DNA double-strand breaks, replication stress, and fork collapse, which is inhibited by mei-41, mus101, and grp to achieve efficient fork progression. The mus101 mutant embryo shows a thin eggshell phenotype due to defects in chorion gene amplification, while the grp mutant has a normal chorion gene copy number in amplification-stage follicle cells. However, the role of Claspin in chorion gene amplification is unknown. This study investigated the functions of Drosophila Claspin during chorion gene amplification and compared them with the functions of mei-41 and mus101 (Choi, 2017).

Drosophila Claspin and mus101 mutant embryos were found to show thin eggshell phenotypes due to reductions in chorion gene amplification, while mei-41 mutant embryos do not show obvious defects in chorion gene amplification. The chorion gene amplification detected by thymidine analog incorporation was greatly affected by Claspin mutations in both initiation- and elongation-stage follicle cells. Although initiation was significantly reduced in the mus101 mutant, the progression of replication forks in the elongation stage was not severely affected. The Claspin protein was enriched in chorion gene amplification foci during the initiation and elongation stages of chorion re-replication in a mei-41-independent manner. These results suggest that Drosophila Claspin and mus101 have a mei-41-independent function in the initiation of chorion gene amplification and Claspin, but not mus101, is important for the efficient progression of replication forks (Choi, 2017).

To understand the biological functions of Drosophila Claspin, this study investigated the basis of the thin eggshell phenotype of Claspin mutants and compared it with that of mus101 and mei-41 mutants. Drosophila Claspin and mus101 were found to be required for the initiation of chorion gene amplification. Claspin, but not mus101, plays a major role in the efficient progression of replication forks. The role of Claspin during amplification was supported by its localization to amplification foci during initiation and elongation. These characteristics were distinct from those of mei-41, suggesting that Drosophila Claspin and mus101 have a unique and mei-41&-independent role in DNA replication during chorion gene amplification (Choi, 2017).

During oogenesis, the mode of DNA replication in somatic follicle cells that encircle germline cells changes from mitotic replication to endoreplication, followed by chorion gene amplification in the absence of genomic DNA replication. Studies of various mutants that show defects in chorion gene amplification have revealed three different phenotypes. In addition to a lack of amplification, some mutants exhibit chorion gene overamplification, and other mutant follicle cells fail to exit the endocycle during the amplification stage and instead perform inappropriate genomic DNA replication throughout the follicle cell genome. These results suggest that distinct signaling pathways exist for the positive and negative regulation of chorion gene amplification and for the repression of genomic DNA replication. In Claspin and mus101 mutant stage 10B follicle cells, neither ectopic genomic replication nor overamplification of the chorion gene was observed. This suggests that Claspin and mus101 are required for chorion gene amplification and that they are not involved in suppressing genomic DNA replication or in negatively regulating chorion gene amplification (Choi, 2017).

The functions of Claspin and TopBP1 in DNA replication are conserved from yeast to mammalian cells and both proteins are important for the initiation of DNA replication. This study found that Drosophila Claspin and mus101 are required for the initiation of chorion gene amplification based on the following observations. First, the intensity of EdU incorporation in follicle cells at the initiation stage and the relative fold amplification of ACE3, which is located 1.5 kb away from the origin, were severely reduced in both mutants. Second, when the EdU double bar was detected in the stage 13 follicle cells of Claspin and mus101 mutants, the length of the bar representing the number of origin firings was significantly shorter than that of the wild type . Lastly, the Claspin protein exhibited a focal localization overlapping with the largest EdU foci known to contain the ORC complex during the initiation stage (Choi, 2017).

In addition to initiation, Claspin affects the replication fork progression rate in mammalian cells (Petermann, 2008) and Mrc1 (yeast Claspin) found in the replisome is essential for rapid replisome progression in vitro (Yeeles, 2017). On the other hand, Dpb11 (yeast TopBP1) is not considered part of the replisome and Xenopus TopBP1 does not seem to be required for the elongation steps of DNA replication. Consistent with these previous reports, this study found that EdU foci were not efficiently resolved into a double bar structure in the Claspin mutant follicle cells at the elongation&-only stage, whereas a significantly higher percentage of mus101 mutant follicle cells exhibited double bar structure formation. Moreover, Claspin staining appeared as a double bar and colocalized with EdU during the elongation stage in follicle cells, visually confirming that Claspin moves along with the replication forks. These results show that Drosophila Claspin and mus101 have conserved functions during chorion gene amplification (Choi, 2017).

Drosophila chorion gene amplification begins with the binding of the ORC complex to replication origins using most of the general DNA replication machinery. Many genes have been reported to affect chorion gene amplification and mutations in most of these genes also result in a loss of ORC foci formation. The exceptions are Myb and dup mutants; normal ORC2 foci have been detected, despite the absence of bromodeoxyuridine foci in the Myb mutant clones and ORC2 foci are smaller in dup mutant follicle cells (Choi, 2017).

This study found that ORC2 localization to amplification loci was significantly reduced in the mus101 K451 mutant compared with the wild type, whereas it was not significantly different in Claspin 45 mutant. Compared with the wild type, the amplification of ACE3 in Claspin 45 and mus101 K451 mutants was reduced to 24.0 and 5.2&-fold relative to actin, respectively. Because ACE3 is the region recognized by ORC2 and where the major ORC2 foci are localized at stage 10B, a significant reduction in ORC2 intensity in the mus101 mutant is likely to result from the reduced copy number of the origin (Choi, 2017).

Additionally, the Dup (Drosophila Cdt1) protein, which usually forms foci at chorion loci, is stabilized and delocalized by various defects in DNA replication, including mus101 K451 mutations. It is not clear if Dup localization is similarly affected in Claspin mutants. Because the size of ORC2 foci is smaller in dup mutant follicle cells than in wild type cells, the reduction in ORC2 intensity found in mus101 K451 mutants may result from the delocalization of Dup. Further analyses will be required to elucidate the detailed molecular events in the initiation steps of chorion gene amplification (Choi, 2017).

A previous study of Drosophila mei-41 RT1 and mus101 D1, a separation-of-function allele that shows defects in the G2/M DNA damage checkpoint, but normal DNA replication, showed that cells lacking these genes are defective in the replication stress checkpoint and exhibit reduced fork progression by 25%-30%, rather than the complete lack of replication. mus101 K451, another separation-of-function allele with the opposite phenotypes, shows defects mostly in the initiation step of chorion gene amplification. Claspin is directly involved in the initiation and elongation steps of chorion gene amplification, although mitotic replication and endoreplication seem to occur normally in both mutants. Because several hypomorphic mutants of pre-RC components also show phenotypic abnormalities only in chorion amplification, amplification may be more sensitive to the activity of the basal DNA replication machinery than to mitotic replication (Choi, 2017).

A recent study reporting the first example of gene amplification in normal mammalian development has identified genes that are selectively amplified in trophoblast giant cells. An investigation into whether the Claspin and TopBP1 play similar functions in mammals will provide useful insights. Drosophila chorion gene amplification will serve as a valuable model for elucidating the mechanism of action of Claspin and mus101 during DNA replication, specific gene amplification, and the replication stress checkpoint (Choi, 2017).

tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription

RNase P is a conserved endonuclease that processes the 5' trailer of tRNA precursors. This study isolated mutations in Rpp30, a subunit of RNase P, and found that these induce complete sterility in Drosophila females. It was shown that sterility is not due to a shortage of mature tRNAs, but that atrophied ovaries result from the activation of several DNA damage checkpoint proteins, including p53, Claspin, and Chk2. Indeed, tRNA processing defects lead to increased replication stress and de-repression of transposable elements in mutant ovaries. Transcription of major piRNA sources collapse in mutant germ cells and that this correlates with a decrease in heterochromatic H3K9me3 marks on the corresponding piRNA-producing loci. These data thus link tRNA processing, DNA replication, and genome defense by small RNAs. This unexpected connection reveals constraints that could shape genome organization during evolution (Molla-Herman, 2015).

Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks

ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, Drosophila lines were generated that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. It was also found that Claspin is phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin is generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, these data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses (Lee, 2012).

Claspin was originally identified in Xenopus laevis egg extracts as a Chk1-interacting protein that was required for DNA replication stress-induced G2 arrest. DNA replication stress induces ATR-dependent phosphorylation of Claspin, which results in a Claspin-Chk1 interaction and phosphorylation and activation of Chk1 by ATR. Claspin protein levels are regulated during the cell cycle, peak at the S/G2 boundary, and are degraded during mitosis. In support of its high expression during S phase, Claspin is reported to have a role during normal DNA replication when in the absence of exogenous DNA damage. Moreover, Claspin is required for terminating DNA damage-induced cell cycle arrest. Phosphorylation of Claspin by Polo-like kinase-1 (Plk1) results in the dissociation of Claspin from chromatin in Xenopus or the degradation of Claspin in human cells, which leads to inactivation of Chk1 and resumption of the cell cycle after prolonged interphase arrest. Most of the work on Claspin has been performed in Xenopus egg extracts and human tissue culture, however, animal models for Claspin have not been reported (Lee, 2012).

To understand the in vivo function of Claspin, mutants of Claspin (CG32251) were generated by imprecise excision of a transposable element. Analysis of the DNA damage checkpoint during early embryogenesis and the larval stage of this mutant showed that Drosophila Claspin is required for cell cycle arrest in response to incompletely replicated DNA. However, Claspin is dispensable for IR-induced cell cycle arrest. Interestingly, Claspin is phosphorylated after IR and HU treatment and a hyperphosphorylated form of Claspin was observed after HU but not after IR treatment. Moreover, the HU-induced hyperphosphorylation of Claspin is attenuated in mei-41/ATR mutant, but not in tefu/ATM mutant. These results suggest that the phosphorylation state and the role of Drosophila Claspin in cell cycle arrest are distinctly regulated by different types of DNA damage: DNA replication stress and DSBs (Lee, 2012).

Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1

The mediator protein Claspin is critical for the activation of the checkpoint kinase Chk1 during checkpoint responses to stalled replication forks. This function involves the Chk1-activating domain (CKAD) of Claspin, which undergoes phosphorylation on multiple conserved sites. These phosphorylations promote binding of Chk1 to Claspin and ensuing activation of Chk1 by ATR. However, despite the importance of this regulatory process, the kinase responsible for these phosphorylations has remained unknown. By using a multifaceted approach, this study found that casein kinase 1 gamma 1 (CK1γ1; Gilgamesh) carries out this function. CK1γ1 phosphorylates the CKAD of Claspin efficiently in vitro, and depletion of CK1γ1 from human cells by small interfering RNA (siRNA) results in dramatically diminished phosphorylation of Claspin. Consequently, the siRNA-treated cells display impaired activation of Chk1 and resultant checkpoint defects. These results indicate that CK1γ1 is a novel component of checkpoint responses that controls the interaction of a key checkpoint effector kinase with its cognate mediator protein (Meng, 2011).

This study sought to resolve the molecular basis of a key step in the checkpoint-dependent activation of Chk1 in response to genomic stress. The activation of Chk1 involves phosphorylation-dependent docking of Chk1 with its cognate mediator protein (Claspin) and recognition of the resulting Claspin-Chk1 complex by ATR-ATRIP. In particular, phosphorylation of Claspin on multiple residues in its CKAD mediates the binding of Chk1. Direct biochemical evidence has indicated the presence of Claspin in this complex enhances the ability of ATR-ATRIP to phosphorylate Chk1 on key residues necessary for the ultimate activation of Chk1. Thus, the kinase that phosphorylates Claspin on the CKAD plays a key role in this pathway (Meng, 2011).

To address the identification of this kinase(s) systematically, a kinome-wide RNAi screen was initially performed in Drosophila S2 cells. Various RNAi screens in these cells have been used to identify numerous regulators in diverse cellular pathways. For the purposes of this study, the fact that Drosophila contains approximately one-third as many kinases as humans was a considerable advantage. S2 cells appear to possess checkpoint-signaling pathways generally similar to those in other metazoan cells. In particular, the Drosophila Chk1 homologue Grapes exhibits a conserved checkpoint function in S2 cells. It functions downstream of Mei-41 (the Drosophila homologue of ATR) and is critical for a proper checkpoint response to genotoxic stress (Meng, 2011).

Nonetheless, the reagents available for the study of checkpoint responses in Drosophila S2 cells are still relatively limited. To circumvent this problem, the Xenopus version of Chk1 was introduced into the Drosophila cells as a more readily traceable marker to monitor the checkpoint response. It was possible to establish a system in which phosphorylation of this reporter could be induced following treatment with a variety of replication inhibitors. Moreover, this response seems to have molecular features similar to those present in vertebrate cells. For example, RNAi-mediated knockdown of Drosophila homologues of ATR and Claspin abolished checkpoint-dependent phosphorylation of the exogenously introduced Chk1 reporter molecule (Meng, 2011).

The results of the screen indicated that knockdown of several casein kinases led to the reduced phosphorylation of Chk1. Following this lead, a candidate-based cDNA overexpression approach was employed to further pinpoint a specific kinase that can directly phosphorylate the CKAD of Claspin. From these tests, it was eventually found that Drosophila Gish, a homologue of CK1γ, could phosphorylate the CKAD of Claspin quite effectively both in vitro and in the Drosophila tissue culture cells. At this juncture, the studies were extended to human cells. Interestingly, humans possess three different versions of CK1γ, namely, CK1γ1, CK1γ2, and CK1γ3. All three proteins have very similar central kinase domains but are significantly different in their N- and C-terminal extensions. It was observed that all three kinases could phosphorylate the CKAD relatively well in vitro. However, further characterizations established that CK1γ1 appears to be the form primarily responsible for the phosphorylation of CKAD in human cells. Upon expression in human cells, only CK1γ1 could phosphorylate the CKAD of Claspin in vivo. More importantly, siRNA-mediated knockdown of CK1γ1 resulted in greatly reduced phosphorylation of Claspin, whereas knockdowns of CK1γ2 and CK1γ3 did not have a significant effect. As expected from the known function of the phosphorylation of Claspin, cells with diminished levels of CK1γ1 were greatly compromised in their ability to carry out the activation of Chk1 in response to a variety of genotoxic agents, including APH, HU, and UV. Furthermore, these cells had the physiological defects characteristic of cells with impairment of the Chk1-mediated signaling pathway. In particular, these cells showed reduced survival following treatment with genotoxic agents, impaired recovery of stalled replication forks, a defective G2/M checkpoint response, and spontaneous DNA damage in the absence of exogenous stress. Taken together, our results indicated CK1γ1 is an important regulator in Chk1-mediated cellular checkpoint responses (Meng, 2011).

The casein kinase 1 family of serine/threonine kinases is highly conserved and ubiquitously expressed. The functions of CK1 encompass a wide variety of processes, including cell proliferation, cell division, apoptosis, circadian rhythms, and others. In mammals, this family contains at least seven members (α, β, γ, ε, γ1, γ2, and γ3) with multiple splicing variants. All of the CK1 proteins share significant homology in the central kinase domain (53%-98% identical), but differ significantly in the flanking N- and C-terminal sequences, which most likely confer unique properties to the various kinases. In the case of CK1γ1, this study identified and isolated multiple splicing variants of this kinase from human U2OS cells. These isoforms contain distinct C-terminal sequences (ranging from 3 to 106 amino acids), display discrete subcellular localizations, and exhibit differences in their abilities to phosphorylate the CKAD. A similar phenomenon has also been observed in various organisms in the case of CK1α, which contains four types of isoforms with distinct localizations and kinase activities. These findings strongly suggest various isoforms of CK1 may participate in distinct cellular activities due to differences in access to and/or affinity for substrates (Meng, 2011).

Interestingly, it was initially difficult to achieve good rescue of CK1γ1-depleted cells with vectors encoding an siRNA-resistant version of the major published form of CK1γ1. However, there are multiple forms of CK1γ1 in human cells. It was possible to clone and express three additional versions of CK1γ1, which were designated as isoforms B, C, and D to distinguish them from the published form (isoform A). In cellular localization studies, it was found that isoform A had the expected prominent localization to cell membranes. However, isoforms B and C resided in both the nucleus and cytoplasm, whereas isoform D was mainly cytoplasmic. In coexpression studies, it was found that isoforms A, B, and C (but not D) could phosphorylate the CKAD of Claspin, although isoforms B and C were more effective than isoform A in this in vivo assay. Ultimately, it was possible to rescue depletion of CK1γ1 by introducing siRNA-resistant forms of isoforms A, B, and C into the cells. It is straightforward to understand how the nuclear isoforms could regulate Claspin, but it is intriguing that a membrane-bound enzyme is also partially responsible. Conceivably, some fraction of this isoform could be absent from the membrane. It is also possible that Claspin could shuttle between the nucleus and cytoplasm and thus be subject to regulation by enzymes in both locations. In this regard, a recent study has shown that perturbed cell-surface signaling through the Sonic Hedgehog (Shh) pathway inhibits ATR-mediated signaling by disrupting the interaction between Claspin and Chk1 (Meng, 2011).

It will be important to understand the mechanisms that control the phosphorylation of Claspin by CK1γ1. It is known that phosphorylation of the CKAD in Xenopus egg extracts is dependent upon ATR. However, ATR itself is unable to phosphorylate the critical sites in the CKAD directly. One possibility would be that ATR might somehow regulate the activity of CK1γ1. However, it has been possible to detect only a subtle increase in the activity of CK1γ1 in response to genomic stress. Moreover, when the only apparent potential target site for ATR (Ser361) was mutated to Ala in CK1γ1, no change was observable in its kinase activity toward the CKAD upon coexpression in U2OS cells. Another possibility is that ATR might regulate the accessibility of Claspin to CK1γ1. Further studies will be required to understand the dynamics of this phosphorylation (Meng, 2011).

In summary, this study identified a conserved casein kinase, Gish/CK1γ1, from Drosophila and humans as a specific enzyme that controls phosphorylation of the CKAD of Claspin. Functional studies have revealed that this kinase is critical for mediating activation of Chk1 and ensuring a proper checkpoint response under conditions of genotoxic stress. Further studies of its regulation and function should help gain more insight into the molecular basis of checkpoint responses (Meng, 2011).


Functions of Claspin orthologs in other species

How the eukaryotic replisome achieves rapid and efficient DNA replication

The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases alpha, delta, and epsilon and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min-1. This sophisticated machine with purified proteins has now been reconstructed, beginning with regulated CMG assembly and activation. Replisome-associated factors Mrc1 (yeast Claspin) and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase epsilon catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase delta can support leading-strand synthesis, but at slower rates. DNA polymerase delta is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase epsilon engagement. It is proposed that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress (Yeeles, 2017).

Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells

Claspin transmits replication stress signal from ATR to Chk1 effector kinase as a mediator. It also plays a role in efficient replication fork progression during normal growth. This study has generated conditional knockout of Claspin and shows that Claspin knockout mice are dead by E12.5 and Claspin knockout mouse embryonic fibroblast (MEF) cells show defect in S phase. Using the mutant cell lines, the crucial roles of the acidic patch (AP) near the C terminus of Claspin in initiation of DNA replication was demonstrated. Cdc7 kinase binds to AP and this binding is required for phosphorylation of Mcm. AP is involved also in intramolecular interaction with a N-terminal segment, masking the DNA-binding domain and a newly identified PIP motif, and Cdc7-mediated phosphorylation reduces the intramolecular interaction. These results suggest a new role of Claspin in initiation of DNA replication during normal S phase through the recruitment of Cdc7 that facilitates phosphorylation of Mcm proteins (Yang, 2016).

Cdc7-dependent and -independent phosphorylation of Claspin in the induction of the DNA replication checkpoint

Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. This study use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. Inhibition of Cdc7 kinase was shown to delay HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. While chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. Claspin is shown to be an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, multiple phosphorylation sites were identified that help to define a Cdc7 phosphorylation motif. Finally, the interaction between Claspin and Cdc7 was shown not to be dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. It is proposed that Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin's function in the DNA replication checkpoint (Rainey, 2013).

Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17

Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. This study shows that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and Rad17 require RPA for association with DNA. Claspin, ATR, and Rad17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork (Lee, 2003).

Chk1 and Claspin potentiate PCNA ubiquitination

Chk1 is a kinase crucial for genomic integrity and an effector of ATR (ATM and Rad3-realated) in DNA damage response. This study shows that Chk1 regulates the DNA damage-induced ubiquitination of proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR (Yang, 2008).

Claspin promotes normal replication fork rates in human cells

The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. This study used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone (Petermann, 2008).

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint

In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. This study shows that in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/CCdh1, with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/CCdh1 reactivation in DNA-damaged G2 cells, Claspin, which is shown in this study to be an APC/CCdh1 substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint (Bassermann, 2008).

Cleavage of claspin by caspase-7 during apoptosis inhibits the Chk1 pathway

Claspin (potential Drosophila homolog: CG32251) is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide causes activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin is cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that each contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress (Clarke, 2005).

Roles of replication fork-interacting and Chk1-activating domains from Claspin in a DNA replication checkpoint response

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. This study shows that Claspin contains a replication fork-interacting domain (RFID, residues 265-605) that associates with Cdc45, DNA polymerase epsilon, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265-331 and 470-600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. This study also find that Claspin contains a small Chk1-activating domain (residues 776-905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, the findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1 (Lee, 2005).

Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase

The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. During this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response (Yoo, 2004).


REFERENCES

Search PubMed for articles about Drosophila Claspin

Bassermann, F., Frescas, D., Guardavaccaro, D., Busino, L., Peschiaroli, A. and Pagano, M. (2008). The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint. Cell 134(2): 256-267. PubMed ID: 18662541

Choi, S. H., Park, J. H., Nguyen, T. T., Shim, H. J. and Song, Y. H. (2017). Initiation of Drosophila chorion gene amplification requires Claspin and mus101, whereas Claspin, but not mus101, plays a major role during elongation.. Dev Dyn [Epub ahead of print]. PubMed ID: 28294450

Clarke, C. A., Bennett, L. N. and Clarke, P. R. (2005). Cleavage of claspin by caspase-7 during apoptosis inhibits the Chk1 pathway. J. Biol. Chem. 280(42): 35337-45. 16123041

Lee, E. M., et al. (2012). Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks. DNA Repair (Amst). 11(9): 741-52. PubMed Citation: 22796626

Lee, J., Kumagai, A., and Dunphy, W. G. (2003). Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol. Cell 11: 329-340. PubMed ID: 12620222

Lee, J., Gold, D. A., Shevchenko, A., Shevchenko, A. and Dunphy, W. G. (2005). Roles of replication fork-interacting and Chk1-activating domains from Claspin in a DNA replication checkpoint response. Mol Biol Cell 16(11): 5269-5282. PubMed ID: 16148040

Meng, Z., Capalbo, L., Glover, D. M. and Dunphy, W. G. (2011). Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1. Mol Biol Cell 22(16): 2834-2847. PubMed ID: 21680713

Molla-Herman, A., Vallés, A.M., Ganem-Elbaz, C., Antoniewski, C. and Huynh, J.R. (2015). tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription. EMBO J 34(24):3009-27. PubMed ID: 26471728

Petermann, E., Helleday, T. and Caldecott, K. W. (2008). Claspin promotes normal replication fork rates in human cells. Mol Biol Cell 19(6): 2373-2378. PubMed ID: 18353973

Rainey, M. D., Harhen, B., Wang, G. N., Murphy, P. V. and Santocanale, C. (2013). Cdc7-dependent and -independent phosphorylation of Claspin in the induction of the DNA replication checkpoint. Cell Cycle 12(10): 1560-1568. PubMed ID: 23598722

Yang, C. C., Suzuki, M., Yamakawa, S., Uno, S., Ishii, A., Yamazaki, S., Fukatsu, R., Fujisawa, R., Sakimura, K., Tsurimoto, T. and Masai, H. (2016). Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells. Nat Commun 7: 12135. PubMed ID: 27401717

Yang, X. H., Shiotani, B., Classon, M. and Zou, L. (2008). Chk1 and Claspin potentiate PCNA ubiquitination. Genes Dev 22(9): 1147-1152. PubMed ID: 18451105

Yeeles, J. T., Janska, A., Early, A. and Diffley, J. F. (2017). How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication. Mol Cell 65(1): 105-116. PubMed ID: 27989442

Yoo, H. Y., Kumagai, A., Shevchenko, A., Shevchenko, A. and Dunphy, W. G. (2004). Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase. Cell 117(5): 575-88. 15163406


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date revised: 10 September 2017

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